• Korean Journal of Microbiology
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• v.51 no.3
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• pp.271-279
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• 2015

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in physiological secretions of mammals. LF shows antibacterial, antiviral and antifungal activities. In the present study, a gene encoding the N-terminal lobe of human lactoferrin (hLF) was isolated, cloned and expressed in methylotrophic yeast, Pichia pastoris. The recombinant hLF-N (rhLF-N) protein was secreted into the culture medium at the level of $458{\mu}g/ml$ in 3 L fermentor. The size of purified hLF-N was estimated as 35 kDa when analyzed by SDS-PAGE and western blotting. The rhLF-N was further confirmed by immunodiffusion using the anti-hLF polyclonal antibody. The expression profile analysis by qRT-PCR showed that the relative mRNA expression of rhLF-N was maximal after 2-3 days of methanol induction and reduced gradually at 4 days. The purified rhLF-N showed broad antibacterial activities against the pathogens such as Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Burkholderia cepacia, and Salmonella typhimurium. However, rhLF-N showed relatively lower activity when compared to peptides derived from LF. In spite of this weak activity, the rhLF-N expressed in P. pastoris might be more advantageous for the industrial application, because rhLF-N is secreted into the culture medium and the production can also be increased by optimization of culture conditions.

• International Journal of Oral Biology
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• v.39 no.3
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• pp.145-151
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• 2014

During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.

• Journal of Plant Biotechnology
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• v.4 no.4
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• pp.143-150
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• 2002

A strong oxidative stress-inducible peroxidase promoter (referred to as SWPA2 promoter) was cloned from tell cultures of sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco cultured cells in terms of biotechnological applications. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the $\beta$-glucuronidase (GUS) reporter gene, the 1314 bp deletion mutant showed approximately 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed following 15 days of subculture compared to other deletion mutants, suggesting that the 1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic cell lines engineered to produce key pharmaceutical proteins. In this respect, we developed transgenic cell lines such as tobacco (Nicotiana tabacum L. BY-2), ginseng (Panax ginseng) and Siberian ginseng (Acanthopanax senticosus) using a SWPA2 promoter to produce a human lactoferrin (hLf) and characterized the hLf production in cultured cells. The hLf production monitored by ELISA analysis in transgenic BY-2 cells was directly increased proportional to cell growth and reached a maximal level (up to 4.3% of total soluble protein) at the stationary phase in suspension cultures. The SWPA2 promoter should result in higher productivity and increased applications of plant cultured cells for the production of high-value recombinant proteins.

• Korean Journal of Agricultural Science
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• v.28 no.2
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• pp.92-98
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• 2001

Lactofenicin is an antibacterial peptide fragment (about 5 kD) derived from lactoferrin (80 kD) that displays the various biological functions. The production of a human lactoferricin (Lactoferricin H) in mouse HC11 mammary epithelial cells was achieved by placing its cDNA under the control of the bovine ${\beta}$-casein gene. To express lactoferricin H in this cell culture system, constructed a hybride-splice signal consisting of bovine ${\beta}$-casein intron I and rabbit ${\beta}$-globin intron II, and a DNA fragment spanning intron 8 of the bovine ${\beta}$-casein gene. Expression of lactofenicin H from this expression vector was identified by RT-PCR, northern and dot blot analysis. RT-PCR using total RNA of HC11 cells transfected with pBL1-cin expression vector yielded a product identified as having a size of the 150bp. Northern blot analysis was identified about 2.3 kb. In dot blot analysis, recombinant lactofenicin H was recognized with anti-human lactofrrnin polyclonal antibody.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.8
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• pp.1175-1182
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• 2017

Objective: To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases. Methods: TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT). Results: The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. Conclusion: This finding signifies the combined use of TALENs and SCNT can generate biallelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.